The present invention concerns heat-sensitive magnetic particles having a predetermined size between 0.05 and 10 xcexcm, which comprises, on the one hand, an internal composite core and, on the other hand, an external layer based on a polymer which is capable of interacting with at least one biological molecule. The invention also concerns production methods and uses of such particles.
In the following account, the term xe2x80x9ccompositexe2x80x9d is synonymous with a magnetic compound which is simple, such as ferrite, or complex, such as ferrite distributed, incorporated or coated in a polymer matrix.
This internal composite core may have a diameter between 20 nm and 10 xcexcm.
Biological diagnosis consists in detecting a disease using the analysis of biological samples (urine, blood, cerebrospinal fluid, sediment, sputum, etc.). In the research context, it is desirable in particular to be able to extract and concentrate, under conditions compatible with the activity assays of certain proteins or nucleic acids, in order to demonstrate the presence, in people who are ill, of a specific protein or DNA or RNA sequence. In the case of proteins, the methods currently used consist in introducing into the medium a large amount of salts which bring about the precipitation of the proteins, and then in separating proteins and salts by centrifugation. However, these methods are very laborious and, in addition, the proteins thus extracted are often denatured, i.e. they have lost their biological properties. This explains the interest which has been shown, in recent years, in magnetic latexes in the biomedical field.
Specifically, the polymer supports in the form of latex are extensively used because they exhibit many advantages, in particular that of offering a large specific surface area (several tens of m2) per gram of particle.
The prior art can be defined by documents EP-A-0,106,873 and EP-A-0,446,260, which describe superparamagnetic monodisperse particles comprising a polystyrene/divinylbenzene copolymer-based porous core into which grains of magnetic iron oxide are incorporated, and a functionalized external layer which is capable of interacting with nucleic acid probes.
According to the method for preparing particles described in these documents, the magnetic iron oxides are incorporated by precipitation of the corresponding salts, which limits the proportion of magnetic filler incorporated, and makes it possible to obtain the magnetic filler only in a surface monolayer, which can induce phenomena of inhibition of biomolecule activity.
Similarly, document EP-A-0,585,868 describes magnetic particles consisting of a core based on a first polymer and on a magnetic layer covering the core consisting of a second polymer in which is distributed the ferrite-based magnetic material, and which is capable of interacting with an antigen or an antibody, the magnetic material being deposited by precipitation of the iron salts.
The incorporated magnetic material is directly exposed to the subsequent treatments of the particles, and a loss of the filler ensues during the use of the particles, which can cause problems in particular of enzymatic inhibition and of denaturation of biological entities.
The subject of document WO-A-94/09368 is the use of gelatin in place of the polymer based on acrylamine derivatives, as we recommend. This document proposes however a crosslinking agent.
Even so, gelatin has functions which are totally different from those of a polymer as claimed. Thus, gelatin is absolutely not heat-sensitive, and it was not at all proven that the crosslinking of heat-sensitive acrylamine derivatives leads to the trapping of the magnetic inclusions present in the internal core.
According to the article by A. Kondo, (A. Kondo, H. Kamura and K. Higashitani (1994) Appl. Microbiol, Biotechnol., 41, 99-105) a method for obtaining magnetic particles is known which comprises a core which is based on a first polymer consisting of a polystyrene, and in which is distributed a magnetic material, and a hydrophilic layer covering the core which is based on a heat-sensitive polymer consisting of poly(N-isoproprylacrylamide). The method described comprises the following two steps. According to a first step for obtaining the magnetic core, the magnetic material is brought into contact with the styrene in the presence of a polymerization initiator. According to a second step for obtaining the hydrophilic layer, the core obtained is brought into contact with N-isoproprylacrylamide and methacrylic acid, in the presence of the above polymerization initiator. Bovine serum albumin is bound to the particles thus obtained in order to subsequently isolate antibodies directed against bovine serum albumin, present in a sample.
This document proposes magnetic particles which comprise inclusions of magnetic material present in the internal core and/or in the external layer. Of course, if said external layer comprises inclusions, they might be released into the reaction medium and prejudice possible applications and uses in the biological and/or diagnostic field requiring specific reactions. Moreover, there is no use of crosslinking agent, and thus no formation of an intermediate layer. If such particles are put together with a solvent, the inclusions will be liberated into the reaction medium, even if these inclusions are present only in the internal core. In addition, said particles are effective only for proteins. The separation of nucleic acids cannot be envisaged.
Thus, to ensure as efficient a separation as possible of these particles in the sample, the applicants have employed thermoflocculation in which the temperature of the sample is increased, this having the effect of completing the action of a magnetic field. It is document WO-A-97/45202 which proposes particles which are superparamagnetic, and which have a very homogeneously distributed magnetic filler, the proportion of which can vary between 1 and 80%, in particular from 25 and 80% by weight with respect to the polymer(s) constituting the particles. The present invention makes it possible to reach high proportions of incorporated magnetic filler, in particular since the method employed makes it possible to distribute the magnetic filler in the form of multilayers. A considerable advantage results therefrom, i.e. the possibility of efficiently separating, from the sample, the particles of the invention, without having to resort to the combined action of another separation technique such as flocculation.
This invention has an objective which is different from that of the present invention. In addition, there is a magnetic internal layer which covers a core which is not magnetic. Moreover, the absence of an intermediate layer is noted. This configuration does not prevent the release of magnetic inclusions from the internal layer into the reaction medium.
While these particles satisfy most of the expectations of the biologists, they comprise a certain number of drawbacks. Thus, these particles do not make it possible to separate, firstly, proteins from nucleic acids, which may be particularly valuable depending on the work undertaken (identification of antibodies, amplification of sequences, etc.) and secondly, nucleic acids. Thus, in the case of nucleic acid separation, the particles according to the invention do not inhibit the various amplification techniques which may be used (PCR, NASBA or TMA). Finally, it is not absolutely necessary to release the extracted substances (proteins or nucleic acids)xe2x80x94with respect to the particles, the identification or amplification assays can be carried out directly after the magnetic separation.